The lipoxygenases are a structurally related family of non-heme iron dioxygenases that function in the production of fatty acid hydroperoxides. Four lipoxygenases have been identified and cloned in humans. Funk, C. D. (1993) Prog. Nuc. Acid Res. Mol. Biol. 45:67-98; Matsumoto et al. (1988) Proc. Natl. Acad. Sci. USA 85: 26-30; Dixon et al. (1988) Proc. Natl. Acad. Sci. USA 85: 416-420; Funk et al. (1990) Proc. Natl. Acad. Sci. USA 87: 5638-5642; Izumi et al. (1990) Proc. Natl. Acad. Sci. USA 87:7477-7481; Yoshimoto et al. (1990) Biochem. Biophys. Res. Comm. 172:1230-1235; Sigal et al. (1988) Biochem. Biophys. Res. Comm. 157:457-464; Brash et al. (1997) Proc. Natl. Acad Sci. USA 94:6148-6152). They oxygenate arachidonic acid in different positions along the carbon chain and form the corresponding 5S-, 12S- or 15S-hydroperoxides (hydro(pero)xyeicosatetraenoic acids, H(P)ETEs). Three of these enzymes are known mainly from the blood cell types in which they are strongly expressed--the 5S-lipoxygenase of leukocytes, the 12S-lipoxygenase of platelets, and the 15S-lipoxygenase of reticulocytes, eosinophils and macrophages. While these are the most widely recognized cellular sources, selective expression is documented in other tissues. For example, both the 12S- and 15S-lipoxygenases are detected in skin. Nugteren et al. (1987) Biochim. Biophys. Acta 921:135-141; Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol. 97:291-297; Takahashi et al. (1993) J. Biol Chem. 268:16443-16448; Hussain et al. (1994) Amer. J. Physiol. 266:C243-C253. The fourth of the known human lipoxygenases, a second type of 15S-lipoxygenase, was cloned from skin and this enzyme is also expressed in prostate, lung, and cornea. Brash et al. (1997) Proc. Nat. Acad Sci. USA 94:6148-6152.
Interest in the biosynthesis of hydroxy derivatives of arachidonic acid in skin stems from the role of essential fatty acids and their derivatives in the structural integrity of normal epidermis (Burr et al. (1929) J. Biol. Chem. 82:345-367; Nugteren et al. (1985) Biochim. Biophys. Acta 834, 429-436; Nugteren et al. (1987) Biochim. Biophys. Acta 921:135-141), and from the potential involvement of arachidonate metabolites in inflammatory and proliferative skin diseases (Hammarstrom et al. (1975) Proc. Natl. Acad. Sci. USA 72:5130-5134; Hussain et al. (1994) Am. J. Physiol. 266:C243-C253; Ziboh, V. A. (1996) Lipids 31: S249-S253). The major products of arachidonic acid metabolism in normal human skin or keratinocytes are 12-hydroxy- and 15-hydroxyeicosatetraenoic acids (12-HETE and 15-HETE) (Nugteren et al. (1987) Biochim. Biophys. Acta 921:135-141; Hammarstrom et al. (1975) Proc. Natl. Acad. Sci. USA 72:5130-5134; Hussain et al. (1994) Am. J. Physiol. 266:C243-C253; Ziboh, V. A. (1996) Lipids 31: S249-S253; Burrall et al. (1988) J. Invest. Dermatol. 4:294-297; Green, F. A. (1989) J. Invest. Dermatol. 93:486-491; Holtzman et al. (1989) J. Clin. Invest. 84:1446-1453; Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol. 97:291-297; Takahashi et al. (1993) J. Biol. Chem. 268:16443-16448).
Biosynthesis of the 15-HETE is better understood in terms of the enzymes involved. It is formed almost exclusively as the 15S enantiomer (Baer et al. (1991) J. Lipid Research 32:341-347; Baer et al. (1993) J. Lipid Research 34:1505-1514.) and its production can be accounted for by the 15S-lipoxygenases present in skin. Nugteren et al. (1987) Biochim. Biophys. Acta 921:135-141; Burrall et al. (1988) J. Invest. Dermatol. 4:294-297; Green, F. A. (1989) J. Invest Dermatol. 93:486-491; Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol. 97:291-297; Takahashi et al. (1993) J. Biol Chem. 268:16443-16448; Baer et al. (1991) J. Lipid Research 32:341-347; Baer et al. (1993) J. Lipid Research 34:1505-1514; Zhao et al. (1995) J. Lipid Res. 36:24444-2449; Brash et al. (1997) Proc. Natl. Acad Sci. USA 94:6148-6152.
Formation of the 12-HETE in human skin is more complex, in that both 12R and 12S enantiomers are produced (Holtzman et al. (1989) J. Clin. Invest. 84:1446-1453; Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol. 97:291-297; Baer et al. (991) J. Lipid Research 32:341-347; Baer et al. (993) J. Lipid Research 34:1505-1514. This is not mainly attributable to autoxidation as the proportions of 12R and 12S vary considerably and, aside from the 15S-HETE, comparable amounts of the other HETE regiosomers are not formed under the usual conditions of in vitro biosynthesis. Formation of the 12S-hydroxy enantiomer can be accounted for by the platelet-type of 12S-lipoxygenase that is a constituent of normal and inflammatory human skin (Hussain et al. (1994) Am. J. Physiol. 266:C243-C253; Takahashi et al. (1993) J. Biol. Chem. 268:16443-16448; Zhao et al. (1995) J. Lipid Res. 36:24444-2449; Brash et al. (1997) Proc. Natl. Acad Sci. USA 94:6148-6152). The enzyme or enzymes involved in the production of the 12R-enantiomer remain uncharacterized.
The first report of 12-HETE in human skin came in 1975, when Hammarstrom et al reported that the involved areas of epidermis in psoriasis have markedly increased concentrations of free arachidonic acid and 12-HETE (Hammarstrom et al. (1975) Proc. Natl. Acad. Sci. USA 72:5130-5134). Chiral analysis of the 12-HETE in psoriasis revealed that the major enantiomer is 12R-HETE (Woollard, P. M. (1986) Biochem. Biophys. Res. Commun. 136:169-175). It was shown subsequently that 12R-HETE is a prominent product in other non-psoriatic proliferative dermatoses (Baer et al. (1995) J. Invest. Dermatol. 104:251-255), and it is also formed in normal human skin as the minor 12-HETE enantiomer (Holtzman et al. (1989) J. Clin. Invest. 84:1446-1453; Baer et al. (1993) J. Lipid Research 34:1505-1514).
It has been questioned whether the enzyme responsible for the 12R-HETE synthesis is a cytochrome P450 or a lipoxygenase. The P450-catalyzed synthesis of 12R-HETE is precedented in rat and human liver microsomes and by purified cytochromes P450, as described in Capdevila et al. (1986) Biochem. Biophys. Res. Commun. 141:1007-1011; Oliw, E. H. (1993) Biochim. Biophys. Acta 1166:258-263; and Bylund et al. (1998) J. Pharmacol. Exp. Ther. 284:51-60. These well-defined P450 reactions are, however, associated with the formation of many additional products that are not typically formed in incubations of skin. The alternative pathway, via a 12R-lipoxygenase, is precedented in a marine invertebrate (Hawkins et al. (1987) J. Biol Chem. 262:7629-7634; Hawkins et al. (1989) FEBS Lett. 247:9-12), but no R-lipoxygenase is known in mammals.
Therefore, what is needed, then, is further characterization of 12-lipoxygenase enzymes in vertebrates, particularly in mammals, and more particularly in humans. A novel isolated and purified 12R-lipoxygenase and a polynucleic acid encoding the same would have broad utility due to its role in arachidonic acid metabolism, a critical metabolic pathway.